Abstract: This study was undertaken in Khartoum State, Sudan, during the period May 2007 to March 2008. A detection system based on real-time PCR has been developed for detection of Escherichia coli strains in patients’ urine. The optimized assay format included two PCR primers. Urine specimens (46) were collected from patients attending different hospitals in Khartoum State. Bacterial DNA was extracted from each urine specimen using the Phenol-Chloroform method. Real time PCR technique was adopted to detect E. coli. The study revealed that 45.7% of the specimens were positive for E. coli. The bacterium was more prevalent in female patients than in male patients. Adult age group was more exposed to the pathogen than the children age group. Real-time PCR technique facilitated detection of E. coli directly in patients’ urine without a need for bacterial culture. The technology could be easily adopted in hospital settings in the Sudan.